Enzymological characterization of recombinant xenopus DG42, a vertebrate hyaluronan synthase.
We have characterized the hyaluronan (HA) synthase activity of the Xenopus DG42 gene product in vitro. The recombinant enzyme produced in yeast does not possess a nascent HA chain and, therefore, is an ideal model system for kinetic studies of the synthase's glycosyltransferase activity. The enzymatic rate was optimal from pH 7.6 to 8.1. Only the authentic sugar nucleotide precursors, UDP-glucuronic acid (UDP-GlcA) and UDP-N-acetylglucosamine (UDP-GlcNAc), were utilized to produce a large molecular weight polymer. UDP-glucose or the galactose epimers of the normal substrates did not substitute. The Michaelis constant, Km, of recombinant DG42 in membranes was 60 +/- 20 and 235 +/- 40 microM for UDP-GlcA and UDP-GlcNAc, respectively, which is comparable to values obtained previously from membranes derived from vertebrate cells. The apparent energy of activation for HA elongation is about 15 kilocalories/mol. DG42 polymerizes HA at average rates of about 80 to 110 monosaccharides/s in vitro. The resulting HA polysaccharide possessed molecular weights spanning 2 x 10(6)-10(7) Da, corresponding to about 10(4) sugar residues. This is the first report characterizing a defined eukaryotic enzyme that can produce a glycosaminoglycan.
Pummill
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PE
Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, USA.
Achyuthan
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AM
DeAngelis
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PL
D000818:Animals; D002413:Cations, Divalent / Q000494:pharmacology; D014453:Glucuronosyltransferase / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D016695:Glycosyltransferases; D000076002:Hyaluronan Synthases; D006820:Hyaluronic Acid / Q000096:biosynthesis; D006863:Hydrogen-Ion Concentration; D008565:Membrane Proteins; D011506:Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D014166:Transferases; D014539:Uridine Diphosphate Sugars / Q000378:metabolism; D029867:Xenopus Proteins; D014982:Xenopus laevis
D002413:Cations, Divalent; D008565:Membrane Proteins; D011506:Proteins; D011994:Recombinant Proteins; D014539:Uridine Diphosphate Sugars; D029867:Xenopus Proteins; D006820:Hyaluronic Acid; D014166:Transferases; D016695:Glycosyltransferases; D014453:Glucuronosyltransferase; C056944:HAS1 protein, Xenopus; D000076002:Hyaluronan Synthases